Real-time optotracing of curli and cellulose in live Salmonella biofilms using luminescent oligothiophenes.

Ferdinand X Choong,K Peter R Nilsson,Keira Melican,Agneta Richter-Dahlfors,Leif Bg Johansson,Sara Fahlén,Mikael Rhen,Marcus Bäck

doi:10.1038/npjbiofilms.2016.24

Ferdinand X Choong, K Peter R Nilsson + Show 6 more

Open Access

https://doi.org/10.1038/npjbiofilms.2016.24

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Abstract

Extracellular matrix (ECM) is the protein- and polysaccharide-rich backbone of bacterial biofilms that provides a defensive barrier in clinical, environmental and industrial settings. Understanding the dynamics of biofilm formation in native environments has been hindered by a lack of research tools. Here we report a method for simultaneous, real-time, in situ detection and differentiation of the Salmonella ECM components curli and cellulose, using non-toxic, luminescent conjugated oligothiophenes (LCOs). These flexible conjugated polymers emit a conformation-dependent fluorescence spectrum, which we use to kinetically define extracellular appearance of curli fibres and cellulose polysaccharides during bacterial growth. The scope of this technique is demonstrated by defining biofilm morphotypes of Salmonella enterica serovars Enteritidis and Typhimurium, and their isogenic mutants in liquid culture and on solid media, and by visualising the ECM components in native biofilms. Our reported use of LCOs across a number of platforms, including intracellular cellulose production in eukaryotic cells and in infected tissues, demonstrates the versatility of this optotracing technology, and its ability to redefine biofilm research.

Highlights

Biofilms are a natural multicellular form of bacterial life, which contribute to resistance against antibiotics, the host immune systems and environmental stresses.[1]
The amyloid curli fimbriae and bacterially produced cellulose have been identified as important extracellular matrix (ECM) components for Escherichia coli, and Salmonella enterica serovars Enteritidis
We evaluated luminescent conjugated oligothiophenes (LCOs) as dynamic optotracers of curli and cellulose during biofilm formation, using spectrophotometric recordings of h-FTAA added to bacterial cultures in the small-volume 96-well format

Summary

INTRODUCTION

Biofilms are a natural multicellular form of bacterial life, which contribute to resistance against antibiotics, the host immune systems and environmental stresses.[1]. Surpassing the conventional amyloid ligands CR and Thioflavin, LCOs identify a broader subset of disease-associated protein aggregates and enable spectroscopic assignment of heterogeneous populations of deposits.[18,19,20,21] The flexible conjugated thiophene backbone distorts in response to non-covalent electrostatic interactions with target molecules. This generates a conformation based, target specific, spectral signature, which in contrast to conventional fluorophores, exhibits an ON/OFF fluorescent signature.[15,16,17] Interactions with amyloid proteins characteristically lead to the flattening of the molecular backbone and a more effective conjugation, causing a red-shift in the fluorescence excitation as well as increased fluorescence emission intensity.[15,16,17] The excitation spectrum in particular is a direct reflection of the LCO backbone geometry. Typhimurium forming biofilm on abiotic surfaces, agar plates, in liquid cultures, intracellularly in eukaryotic cells, and in mouse liver

RESULTS

DISCUSSION